Recent advances in gene transfer technology have opened up new possibilities for introducing desirable traits into plants. A number of such genes have been introduced, in order to confer upon the host plant some measure of protection against environmental stresses. Examples include genes conferring tolerance to chemical herbicides such as glyphosate (Comai, Nature, 317, 741-744 (1985) and Shah, Science, 233, 478-481 (1986)), phosphinothricin (De Block, EMBO, 6, 2513-2518 (1987)), bromoxynil (Stalker, 1987, International Patent Application No. PCT/US87/00044), and sulfonylureas (Haughn, Mol. Gen. Genet., 211, 266-271 (1988)). Transgenic plants have also been engineered to resist certain insect pests (Adang, 1985, published European Patent Application No. 142,924 and Vaeck, Nature, 328, 33-37 (1987)), fungal diseases (Taylor, Mol. Gen. Genet., 210, 572-577 (1987)), and viral diseases (Abel, Science, 232, 738-743 (1986) and Nelson, Bio/Technol., 6, 403-405 (1988)).
Another area of interest is the design of plants, especially crop plants, with added value traits. An example of such a trait is improved nutritional quality in food crops. Lysine, an amino acid essential in the diet of humans and monogastric animals, is among the three most limiting nutrients in most of the cereal crops. Consequently, grain-based diets, such as those based on corn, barley, wheat, rice, maize, millet, sorghum, and the like, must be supplemented with more expensive synthetic lysine or with lysine-containing oilseed protein meals. Increasing the lysine content of these grains or of any of the feed component crops would result in significant added value. To date, attempts to elevate lysine levels in plants have relied on conventional breeding methods and, more recently, mutagenesis and cell culture technology.
Naturally-occurring high lysine mutants of maize (Mertz, Science, 145, 279 (1964) and Nelson, Science, 150, 1469-1470 (1965)), barley (Munck, Science, 168, 985-987 (1970)), and grain sorghum (Singh et al., Crop Sci., 13, 535-539 (1973)) have been identified. In each case, the improved lysine content results not from increased free lysine production, but from shifts in the overall protein profile of the grain: the reduced levels of lysine-deficient endosperm proteins (prolamines) are complemented by elevated levels of more lysine-rich proteins (albumins, globulins and glutelins). While nutritionally superior, these mutants are associated with reduced yields and poor grain quality, limiting their agronomic usefulness.
An alternative approach used to improve nutritional quality has been in vitro selection for biochemical variants having elevated free lysine pools. Lysine is a member of the "aspartate family" of amino acids in higher plants and microorganisms (see FIG. 1). As such, the regulation of its biosynthesis is intimately connected to that of the other members of the family: threonine, methionine and isoleucine. Regulation of metabolite flow appears to be chiefly through endproduct feedback inhibition at key enzymatic steps. The first of these steps, the phosphorylation of aspartate catalyzed by aspartate kinase (AK), is common to all four endproducts. A second site of regulation is at the branch-point reaction: the condensation of pyruvate with aspartyl semialdehyde to form dihydrodipicolinic acid. This reaction is the first one unique to the biosynthesis of lysine and is catalyzed by dihydrodipicolinic acid synthase (DHDPS), an enzyme shown to be strongly feedback inhibited by lysine in plants where it has been examined (Wallsgrove et al., Phytochem., 20, 2651-2655 (1981), and Kumpaisal, Plant Physiol., 85, 145-151 (1987)).
There is evidence to suggest the existence of more than one form of AK (Miflin, 1980, in The Biochemistry of Plants, vol. 5, Amino Acids and Derivatives, Stumpf and Conn (eds.) pp 420-426, Academic Press). One form is sensitive to inhibition by threonine, the other to inhibition by lysine. The growth of plant cell cultures is inhibited in the presence of equimolar amounts of lysine plus threonine. This inhibition may be reversed by the addition of methionine or homoserine (which may be readily converted to methionine) (Green et al., Crop Sci., 14, 827-830 (1974)). Hibberd Planta, 148, 183-187 (1980)) selected stable lines of maize callus that were resistant to this growth inhibition. These lines overproduced threonine (6-9 fold) and to a lesser extent, methionine, lysine and isoleucine (2-4 fold). There was evidence that a lysine-tolerant AK was responsible for the changes observed. In the lines that were regenerated to whole, fertile plants, the overproduction was a stable, heritable trait (Hibberd et al., PNAS, 79, 559-563 (1982 )). Similar selections have been carried out on tobacco (Bourgin, 1982, in Variability in Plants Regenerated from Tissue Culture, Earle and Demarly (eds.), pp 163-174, Praeger, New York), barley (Arruda, Plant Physiol., 76, 442-446 (1984)), and carrot (Cattoir-Reynaerts, Biochem Physiol. Pfanzen, 178, 81-90 (1983)).
Lysine analogs, in particular S(2-aminoethyl)cysteine (AEC) have also been used either alone or in conjunction with lysine plus threonine selections in attempts to isolate lysine-overproducing mutants. Sano et al. (J. Gen. Appl. Microbiol., 16, 373-391 (1970)) were able to isolate high lysine bacterial mutants using AEC selection and AEC was proposed to act as a false feedback inhibitor of AK or DHDPS or both. Attempts to isolate similar mutants in plants have had mixed results. Widholm (Can. J. Bot., 54, 1523-1529 (1976) mutagenized tobacco suspension cells and selected AEC-resistant cell lines that overproduced lysine by ten-fold. Pearl millet mutants were isolated that overproduced lysine by 5-7 fold (Boyes et al., Plant Sci., 50, 195-203 (1987)). Bright (Planta, 146, 629-633 (1979)) selected AEC-resistant barley lines that did not accumulate lysine in the absence of AEC and were shown to be AEC uptake mutants. There was also evidence that AEC exerted its inhibitory effects by being incorporated into proteins rather than by interfering with lysine biosynthesis. Schaeffer et al. (Plant Physiol., 84, 509-515 (1987)) applied sequential AEC and lysine plus threonine selections to obtain race mutants that had 14% higher lysine in seed storage proteins, but not higher free lysine. An AEC-resistant potato culture was selected by Jacobsen (J. Plant Physiol., 123, 307-315 (1986)). This mutant had higher overall amino acid levels than control cultures but this was not due to overproduction of lysine, threonine or methionine. Negrutiu (Theor. Appl. Genet., 68, 11-20 (1984)) subjected tobacco protoplasts to mutagenesis followed by AEC selection. Two resistant cell lines were obtained that overproduced lysine by 10-30 fold. Biochemical and genetic analysis revealed a feedback-insensitive DHDP synthase. The trait was inherited as a single dominant gene.
Heretofore, recombinant DNA and gene transfer technologies have not been applied to the area of increased metabolite production for added value in plants. However, it is known that the bacterium Escherichia coli synthesizes lysine by a pathway essentially identical to that of higher plants. Dihydrodipicolinic acid synthase is encoded by the dap A gene of E. coli and, while sensitive to lysine (Yugari et al., Biochim. Biophys. Acta, 62, 612-614 (1962)), it is at least 200-fold less sensitive to inhibition by lysine in vitro when compared to the same enzyme isolated from plants. Further, E. coli cells carrying the dap A gene on a multicopy plasmid accumulate high levels of free lysine (Dauce-LeReverand, Eur. J. Appl. Microbiol. Biotechnol., 15, 227-231 (1982)), suggesting that DHDPS catalyzes a rate-limiting step. The gene has been sequenced and characterized (Richaud, J. Bacteriol., 166, 297-300 (1986)) and Glassman, 1988, M.S. Thesis, University of Minnesota, Minneapolis, MN).
Considering the relative inability of conventional breeding and tissue culture technology to readily obtain plants accumulating significantly higher levels of lysine, a need exists to apply recombinant DNA and gene transfer technology to produce such plants.